This invention relates generally to acyl derivatives of deoxyribonucleosides and to the use of those derivatives to enhance the delivery of exogenous deoxyribonucleosides to animal tissue. More specifically, this invention relates to the acyl derivatives of 2xe2x80x2-deoxyadenosine, 2xe2x80x2-deoxyguanosine, 2xe2x80x2-deoxycytidine and 2xe2x80x2-deoxythymidine and the use of those novel derivatives to increase the bioavailability of the deoxyribonucleosides to animal tissue and thereby to support cellular metabolic functions. Even more specifically, this invention relates to the use of the novel acyl derivatives to treat or prevent a variety of physiological and pathological conditions in cell tissue, including damage by radiation, sunlight, mutagens, wounds, and other conditions.
There are many physiological and pathological conditions of animal tissue where the supply of exogenous deoxyribonucleosides may have useful therapeutic applications. In the treatment of wounds, repair of liver tissue, promotion of repair and survival after radiation, and numerous other conditions, the supply of DNA and/or deoxyribonucleosides at a high and sustained level may substantially improve the natural DNA and tissue repair processes of the affected cells.
In promoting wound healing, liver regeneration, recovery from radiation damage, and in other pathological and physiological conditions, it is likely that exogenously supplied DNA serves merely as a storage depot for deoxyribonucleosides. That depot gradually releases deoxyribonucleotides and deoxyribonucleosides during enzymatic degradation. Thus the administration of deoxyribonucleosides or derivatives disclosed herein may have value as a method for delivering those deoxyribonucleosides to tissues, which method is preferable to the administration of foreign DNA insofar as wound healing, tissue regeneration, recovery from irradiation, and the like, is concerned.
A number of investigators have attempted to use DNA and/or deoxyribonucleosides to treat a variety of conditions in experimental animals and to enhance or augment cellular repair processes, including DNA repair. It has been demonstrated that administration of exogenous DNA to experimental animals after exposure to ionizing radiation can result in dramatically increased survival and functional recovery. Studies on cell cultures in vitro demonstrate that the actual restorative agents are probably deoxyribonucleosides, the enzymatic degradation products of DNA. These compounds enhance the actual repair of damaged DNA in vitro. However, depolymerized DNA or deoxyribonucleosides administered to animals were ineffective in promoting survival or recovery after irradiation. Kanazir et al., Bull. Inst. Nuc. Sci xe2x80x9cBoris, Kidrinchxe2x80x9d 9:145-153 (1959). There is reason to believe that this apparent contradiction is due to the rapid catabolism of deoxyribonucleosides in vivo by the liver and other organs. Thus, after administration of deoxyribonucleosides, tissues were only exposed to effective concentrations for a matter of minutes. Beltz, et al., Bioch. Biophys. Acta 297:258-267 (1973). In cell cultures, optimum survival after irradiation was found when deoxyribonucleosides were present in the incubation medium for at least 3 hours. When DNA is administered by intraperitoneal injection, it is gradually depolymerized to give a sustained release of free deoxyribonucleosides into the circulation. DNA is not, however, a suitable pharmaceutical agent to administer to humans, either orally or parenterally.
Hunting, D. J., et al., Carcinogenesis 6:1525-1528 (1985), disclose that deoxyribonucleotide synthesis is rate limiting for excision repair of UV-induced DNA damage. The authors found that there was an increase in repair ligation in cells made permeable to added deoxyribonucleotide triphosphates.
Golba, S., et al., Int. J. Rad. Biol. 13:261-268 (1967), disclose that after whole-body irradiation, administration of heterologous DNA improved survival and accelerated the rate of recovery of body weight and of red blood cells, granulocytes and lymphocyte counts in the peripheral blood. No secondary disease or change in the blood count was observed in the next 12 months. Goh, K., Proc. Soc. Exp. Biol. Med. 145:938-943 (1974), discloses addition of exogenous deoxyribonucleotides resulted in prevention or healing of xe2x80x9cpulverizedxe2x80x9d chromosomes found in cultures of leukocytes taken from a human subjected to accidental exposure to fast neutron and gamma irradiation. Horikawa, M., et al., Exp. Cell Res. 34:198-200 (1964), disclose the effect of the addition of various cell extracts and compounds to an incubation medium containing mouse L cells in culture which were irradiated in culture with X-irradiation (2000 R). Homogenates of L cells, L cell nuclei, or purified DNA from either L cells or salmon sperm all strongly enhanced the survival of the irradiated cells. RNA from either yeast or L cells was found to be ineffective. The authors suggest that the DNA hydrolysates (e.g., deoxynucleotides) are the actual reactivating agents, since heterologous DNA is as effective as homologous DNA.
Pantic, V., et al., Nature 193:993-994 (1962), disclose administration of DNA to X-irradiated rats given lethal doses of radiation. The authors found that while DNA treatment did not totally prevent cellular damage in the intestine and liver after irradiation, tissue structure and function were much closer to normal in DNA-treated animals examined 4 or 9 days after irradiation than in untreated irradiated controls.
Paoletti, C., et al., Rev. Francais. Etudes Clin. et Bio. 9:950-955 (1964), disclose a study on the effect of administration of DNA and 2-aminoethyl-isothiouronium (AET) to rats. Mice were given a mixture of AET and thiogel orally, then irradiated (700 rad) and subsequently given i.p. injections of 1 mg calf thymus DNA. The mice receiving the DNA injections recovered their weight and initial leukocyte counts more rapidly than mice similarly treated but not receiving the DNA injections.
Petrovic, D., et al., Int. J. Radiat. Biol. 18:243-258 (1970), disclose evidence concerning the molecular basis of the restorative effect of DNA in cultured mammalian cells. The authors found that the survival of irradiated cells in culture was enhanced by the addition of either DNA or equimolar amounts of deoxyribonucleosides. DNA was effective only if serum containing active deoxyribonuclease was present in the incubation medium. Thus, the authors concluded that the deoxyribonucleosides were probably the actual reactivating factors responsible for repair of radiation-induced damage. In another study, Petrovic disclosed that maximal restoration is attained when deoxyribnucleosides are in the incubation medium for at least 3 hours after irradiation. The best restoration was achieved with either a mixture of all four major deoxyribonucleosides, or a combination of deoxyguanosine with either deoxyadenosine or deoxycytidine. Petrovic, D., et al., Studia Biophysica 43:13-18 (1974). Petrovic et al. also report that in irradiated HeLa cells, treatment with a mixture of the four major deoxyribonucleosides increased survival. Petrovic et al., Int. J. Radiat. Res. 11:609-611 (1967).
Savkovic, N., Nature 203:1297-1298 (1964), discloses that 8 or 17 day old rats subjected to X-radiation (600 rem), and immediately treated with homologous testes DNA, had a much higher fertility rate than did untreated irradiated controls. Histological studies demonstrated that DNA treatment after irradiation markedly protected the structural integrity of the testes and the function of the spermatogenic processes. Savkovic also reported that heterologous DNA extracted from various organs of adult rats was effective in enhancing the survival of mice subjected to irradiation. The DNA reduced the effects of radiation by a factor of 9 to 13. Savkovic, N., et al., Nature 211:1179-1180 (1966). Savkovic, N., et al., Int. J. Rad. Biol. 9:361-368 (1965) also disclose that treatment of irradiated rats with homologous DNA, isolated from liver, thymus and spleen, increased survival and fertility of the survivors. The death rate of the progeny of the irradiated rats was strongly reduced in the case of animals that received DNA after irradiation.
In another study, exposure of cultured calf liver cells to X-radiation was found to cause chromosomal damage. When cells were incubated with either DNA or equimolar concentration of deoxyribonucleotides after irradiation, there was a marked reduction in the incidence of chromosome damage. A mixture of dAMP and dGMP was as effective as a mixture of all four major deoxyribonucleotides. Ribonucleotides were ineffective in preventing radiation-induced chromosome damage. Smets, L. A., et al., Int. J. Rad. Biol. 13:269-273 (1967).
In a related study, administration of dCMP or dTMP to irradiated mice was found to improve the restoration of hematopoietic function. Soska, J., et al., Folia Biologica 5:190-198 (1959).
In another study of mice irradiated with gamma radiation, administration of either a yeast RNA hydrolysate, an equimolar mixture of 3xe2x80x2-nucleotides or a mixture of nucleosides resulted in a significant prolongation of life span. However, long-term survival was not enhanced. The nucleic acid derivatives were administered 30 minutes, 2 days, and 4 days after irradiation. The author observed that the nucleosides, nucleotides, and RNA hydrolysate did not increase the number of surviving stem cell colonies in spleen or bone marrow, but rather appeared to improve the functional capacity of irradiated cells during the critical period after irradiation. These compounds also appeared to accelerate the process of maturation and differentiation of the progeny of surviving stem cells. Sugahara, T., et al., Brookhaven Symposia in Biology, 284-302 (1967).
In a study of guinea pigs subjected to X-radiation, animals given RNA or ATP immediately before and after irradiation had much higher 21-day survival rates than did untreated irradiated controls. Most of the animals that survived the 21-day observation period recovered fully, with no secondary radiation-induced disease. Wagner, R., Int. J. Rad. Biol. 12:101-112 (1967).
In another study, administration of DNA from different sources, including calf thymus, rat liver and spleen, herring and salmon sperm, and Ehrlich ascites carcinoma cells was studied in rats given lethal doses of gamma irradiation. All forms of DNA significantly increased the survival of the irradiated rats. The quantitative differences in the effects of the DNA from different sources were directly related to the molecular weight. The authors found a reduction in therapeutic efficiency which is proportional to the reduction in molecular size upon DNA shearing. Wilczok, T., et al., Int. J. Rad. Biol. 9:201-211 (1965).
The incidence of chromosomal abnormalities in lymphocytes from radiologists chronically exposed to X-rays, was determined before, during, and after treatment with DNA and ATP. The basal incidence of chromosomal damage was substantially higher than in unexposed control subjects. Daily injection of DNA and ATP resulted in 2 to 3 fold decreases in the frequency of chromosomal abnormalities. Following discontinuation of treatment, the incidence of chromosomal damage returned toward pretreatment levels. (Goyanes-Villaescusa, Lancet II:575 (1973).
There have also been reports on the use of DNA preparations to treat wounds. For example, Dumont, Ann. Surg. 150:799 (1959), disclose that exogenous DNA, applied to experimental wounds in rabbit ears, accelerated the growth of granulation tissue in the wounds. A mixture of DNA plus deoxyribonuclease (the enzyme primarily responsible for degradation of DNA) was more effective in accelerating fibroplasia than either DNA or deoxyribonuclease alone. The total amount of granulation tissue formed after treatment with DNA was not greater than in untreated controls; the onset and rate of its growth were however significantly accelerated. The authors suggest that low polymer DNA fragments are the actual active agents.
Nicolau et al., Der Hautartzt 17:512 (1966), disclose a study on experimental skin wounds on the backs of rats which were treated daily with a It solution of DNA in physiological saline. The wounds treated with local application of DNA were cicatrized within four to eight days; those treated only with physiological saline were cicatrized only after 10 to 15 days.
Marshak et al., Proc. Soc. Exp. Biol. Med. 58:62 (1945), disclose that application of DNA to experimental skin wounds in rats resulted in a significant acceleration of the growth of granulation tissue within the wounds, as compared to untreated controls. Although the granulation tissue appeared sooner in treated wounds, the final amount of granulation tissue was not abnormal.
Newman et al., Am. J. Physiol. 164:251 (1951), disclose a study of rats subjected to partial hepatectomy. The course of liver regeneration was followed for 11 days. The livers of rats treated with DNA regenerated significantly faster than livers in untreated animals. RNA treatment also accelerated liver regeneration, though not as markedly as DNA-administration.
Certain derivatives of deoxyribonucleosides have been prepared. Casida et al., Biochemical Pharmacology vol. 15, p. 627-644, 1966, describe the preparation of the 3xe2x80x25xe2x80x2-diacetyl, dipropionyl and dibutyryl esters of 2xe2x80x2-deoxythymidine. Rosowsky et al., Cancer Treatment Reports vol. 65 No.1-2, p. 93-99, January/February 1981, and Ensminger et al., Biochemical Pharmacology vol. 28, p. 1541-1545, October 1978, describe the use of thymidine 5xe2x80x2-O-pivaloate to supply thymidine to tissues.
Since the primary determinant of recovery or survival after exposure to ionizing radiation or chemical mutagens is the preservation or repair of DNA, a number of compounds have been found which, when present in an organism at the time of exposure to radiation or chemical mutagens, attenuate the damage to DNA and other cellular structures. Included in this class of compounds are antioxidants, sulfhydryl compounds, and the enzymes superoxide dismutase and catalase. However, these compounds have been found to be only moderately protective or practical to use in vivo, in part because they can be toxic in effective concentrations. Since these compounds must be present in the organism at the time of exposure to radiation or chemical mutagens, they are obviously not useful in the case of unexpected or accidental exposure.
Reportedly, sulfhydryl compounds are the most effective radioprotective agents known. Examples of these compounds include mercaptoethylamine (MEA), 2-xcex2-amino-ethyl-isothiouronium-Brxe2x80x94HBr (AET), 5-hydroxytryptamine (HT), and 5-2-(3-amino-propylamino)ethylphosphorothiotoic acid (WR-2721). However, many of these compounds are toxic. Thus, several investigators have attempted to increase protection against radiation damage and to decrease toxicity by using mixtures of these chemical protectors. The results of these studies demonstrate that the administration of mixtures of radioprotectors not only increases the degree of protection for short and long term survival compared with that from each substance given separately, but also diminishes the toxicity of compounds such as AET or MEA. Administration of sulfhydryl chemical radioprotectors before exposure to radiation diminishes markedly the changes induced by radiation in the structures. Maisin, J. R., in: Symposium on Perspectives in Radioprotection, Armed Forces Radiobiology Research Institute, Bethesda, Md., p. 53 (1987).
Thiols reportedly protect DNA by mechanisms comprising hydroxyl radical scavenging and DNA radical repair mechanisms. Thus, the extent of interactions of thiols with DNA determines the amount of protection. Cationic thiols (2-[(aminopropyl)amino]ethanethiol (WR-1065) and cysteamine) are better protectors than neutral thiol (2-mercaptoethanol and dithiothreitol) which are in turn better protectors than anionic thiols (glutathione (GSH), 2-mercaptoethanesulfonic acid, and mercaptosuccinate). Such differential binding provides a basis for understanding why WR-1065, which scavenges hydroxyl radicals at a rate comparable to that for GSH, effectively protects cells at concentrations well below those of GSH. Fahery, R. C., ibid., p. 31.
In studies of Chinese hamster V-79 cells treated with gamma radiation and with bromodeoxyuridine (BrdUrd) and light photolysis were compared. When treated with gamma radiation, WR-2721 was found to improve cell survival both by acting as a reducer of gamma radiation, and by causing increase in DNA repair and increase in rejoining of DNA strand breaks. Cysteamine has been shown to act as a reducer of gamma radiation damage without affecting the rejoining of strand breaks or DNA repair capacity. Nicotinamide (NA) has been shown to directly affect DNA repair through the polyADPribose system which is activated by DNA single strand breaks, thus providing NA concentration dependent protection or sensitization. These compounds exhibit a different effect on cells treated with BrdUrd and light compared with gamma radiation. WR-2721 does not reduce strand break formation. MEA and NA reduce damage formation by about 30%. WR-2721 did not affect the rejoining of BrdUrd/light-induced DNA strand breaks. Only NA increased the repair capability of cells subjected to DrdUrd and light damage. Prager, A., et al., ibid., p. 43.
In addition to the use of-thiols, radioprotection has been achieved with xe2x80x9cbiological response modifiersxe2x80x9d (BRM), either alone or in combination with other agents. Such biological response modifiers include glucan, OK-432, Biostim, PSK, Lentinan, Schizophyllan, Rhodexman, Levan, Mannozym, and MVE-2. Of these BRM""s, glucan was found to be the most radioprotective. Glucan is a beta 1-3 polyglycan isolated from the yeast Saccharomyces cerevisiae. Glucan""s radioprotective capacity is attributed to its ability both to protect and/or enhance recovery of hemoatopoietic stem cell populations, and to enhance or maintain the function of macrophage cell populations important in combatting otherwise lethal post-irradiation opportunistic infections. The combination of glucan and WR-2721 resulted in both additive and synergistic radioprotective effects. Patchen, M. L., ibid., P. 68.
Other polysaccharides have also been found to be radioprotective. Intravenous administration of the polysaccharide extracted from the yeast Rhodotorula rubra, mannane mannozyme (MMZ), and the particulate polyglucans GLP/B04 and GLP/B05 (unbranched glucans with alternating B-1,3 and B-1,6 bonds), significantly decreased the mortality of mice exposed to a single dose of X-rays. Maisin, J. R., ibid., p. 69.
The cytokines IL-2 and TNF have also been found to be effective radioprotective compounds. Cytokines are released upon administration of numerous inflammatory agents. Many of these inflammatory agents stimulate the reticuloendothelial system and are radioprotective. Neta, R., ibid., p. 71.
Thymic peptides, such as thymic factor TP-5, have also been reported to reverse or greatly ameliorate immune depression due to limited portal irradiation of thymus, circulating blood, and lymphoid tissues. The immune restorative effect of thymic factors is due to their maturational effect on bone marrow immunocyte precursors. Chretien, P. B., ibid., p. 72.
The antioxidant enzymes glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), catalase, glutathione reductase and glutathione transferase scavenge free radical species produced by radiation and/or the products of free radical cellular damage, and thus play a role in radioprotection. GSH-Px exhibits and best correlation between enzyme activity and cell radiosensitivity. Administration of enzyme preparations or drugs or chemicals which mimic or activate or induce these enzymes may enhance radioprotection. The radioprotectors MEA, WR-2721 and diethyldithiocarbamate (DDC) enhance mouse liver GSH-Px activity 1 to 2 hours after administration. Selenium and selenium-containing compounds also exhibit a small radioprotective effect. The levels of GSH-Px in mouse bone marrow were found to increase 30% 24 hours after administration of selenium. When selenium was administered before WR-2721, a decrease in toxicity and an increase in radioprotection was observed. Superoxide dismutase (SOD) and catalase were also observed to increase upon administration of selenium. In addition, metal ions and metal-containing compounds which mimic antioxidant enzymes may also act as radioprotectors. Copper and zinc metal ions in SOD are marginally radioprotective. Mimetics of SOD include bis (3,5-diisopropylsalicylato) copper and the bivalent copper complex of 3-mercapto-2-hydroxypropylether of dextran. Dumar, K. S., ibid., p. 89. For a review on SOD, see Fridovich, I., Annu. Rev. Biochem. 44: 147-159 (1975).
Induction of metallothionein (MT) in the body by treatment with some heavy metals or immunostimulants has been found to be a potent means for inducing radioprotection. The metal salts CdCl2, MnCl2 or zinc acetate or the immunostimulants OK-432 or IL-1 elevates MT levels in the liver of pretreated mice 10 to 20 times of the control level. The number of leukocytes as well as erythrocytes were reduced temporarily even in pretreated mice. However, the cell counts of pretreated mice showed a faster recovery. Matsubara, J., et al., ibid., p. 99.
Vitamin A and beta carotene have also been suggested as radioprotective agents. They may be involved, in ameliorating the oxidative damage in tissues of irradiated mammals which results from production of free radicals such as hydroxy radicals or H2O+ and its daughter products. Radiation may also create injury to cell structures either by the direct effect of radiation or by the production of toxic metabolites. Radiation injury results in disturbed extracellular and intracellular oxygen levels and perturbed intracellular electron transport and metabolism. Oxidative damage may be enhanced by sudden elevations of local oxygen levels caused by reperfusion of tissues after radiation-induced vasoconstriction or by reversal of radiation-induced bronchoconstriction. Damage occurs where local oxygen levels are in excess of what the tissues can consume. Vitamin A and beta carotene were found to exhibit protective action in rodents exposed to whole body and local radiation. Seifter, E., et al., ibid. p. 104.
Prostaglandins and related compounds of the arachidonic acid cascade protect cells in vivo from some degree of ionizing radiation injury. Among the array of physiological actions of prostaglandins is the protection of cells and tissues from a variety of injuries including strong acids, bases, and absolute ethanol. Prostaglandins were found to exhibit maximal protection at levels a thousandfold lower than those needed for WR-2721. Hanson, W. R., ibid., p. 105.
Methylene blue, a compound used clinically as an anti-inflammatory, antimalarial, and antibacterial agent as well as in the treatment of carbon monoxide poisoning and as an antidote for cyanide poisoning, as also found to protect the intestinal mucosa of rats subjected to sublethal radiation-induced damage. Irradiation damages tissues through the production of highly bioactive free radical species. Therefore, it was hypothesized that methylene blue would also protect irradiated rats from free-radical mediated tissue damage. Scheving, L. E., et al., ibid., p. 115.
In addition, N-arylacetyldehydroalanines reportedly inhibit superoxide anion and hydroxyl free radical-mediated processes, thereby providing radioprotective activity. Buc-Calderone, P., et al., ibid., p. 116.
While the strategy of delivering DNA and/or deoxyribonucleosides to physiologically or pathologically damaged tissue has been recognized, the art has heretofore failed to provide satisfactory methods for introducing deoxyribonucleosides in sufficiently high and reliable amounts to successfully treat the pathological and physiological conditions and to promote cellular repair and survival of the animal. Moreover, although a variety of compounds have been developed which protect animals against some effects of ionizing radiation or chemical mutagens, deoxyribonucleosides provided to tissues for a sufficient time have the greatest clinical potential for post-exposure treatment of such damage. Clinical implementation of this strategy, however, awaits development of satisfactory and convenient methods for delivering adequate quantities of deoxyribunucleosides to tissues in vivo. Similarly, full appreciation and clinical implementation of the capacity of deoxyribonucleosides to promote wound healing or tissue repair awaits development of satisfactory methods for their delivery to tissues in vivo.
It is thus a primary object of this invention to identify pharmaceutically acceptable compounds which can efficiently be used to deliver pharmacologically effective amounts of deoxyribonucleosides or their respective derivatives to animal tissue.
It is still a further object of this invention to provide a family of deoxyribonucleoside derivatives which can be effectively administered orally or parenterally, which have no undesirable toxic effects, and which can be administered to animals and humans to effectively promote cellular repair in a number of physiological and pathological conditions and to promote survival of the animal when administered after exposure to radiation has occurred.
It is still a further and related object of this invention to provide certain derivatives of 2xe2x80x2-deoxyadenosine, 2xe2x80x2-deoxyguanosine, 2xe2x80x2-deoxycytidine, and 2xe2x80x2-deoxythymidine which, when administered to an animal, enhance the bioavailability of those deoxyribonucleosides to the animal tissue.
It is a related object of this invention to substantially improve the bioavailability of 2xe2x80x2-deoxyadenosine, 2xe2x80x2-deoxyguanosine, 2xe2x80x2-deoxycytidine, and 2xe2x80x2-deoxythymidine by enhancing the transport of these deoxyribonucleosides across the gastrointestinal tract, the blood-brain barrier, and other biological membranes.
It is still a further and more specific object of this invention to provide a family of deoxyribonucleoside derivatives for the treatment of a variety of heart, muscle, liver, bone, skin, and other pathological and physiological conditions.
It is still a further object of this invention to provide deoxyribonucleoside derivatives and methods for using those derivatives which are safe, inexpensive, and which accelerate the normal cellular processes of regeneration and healing.
These and other objects of the invention are achieved by the administration of certain acyl derivatives of 2xe2x80x2-deoxyadenosine, 2xe2x80x2-deoxyguanosine, 2xe2x80x2-deoxycytidine, and 2xe2x80x2-deoxythymidine. These acyl derivatives can be used to prevent or treat radiation, sunlight and mutagen-induced cellular damage, to improve the healing of wounds, or repair damaged tissues, and in the treatment of other physiological and pathological tissue conditions.
Broadly, the acyl derivatives of 2xe2x80x2-deoxyadenosine are those having the formula 
wherein R is hydrogen or an acyl radical of a metabolite other than acetyl, with the proviso that at least one R is not hydrogen, or the pharmaceutically acceptable salt thereof.
The preferred acyl derivatives of 2xe2x80x2-deoxyadenosine are those having the formula 
where R is H or an acyl group derived from a carboxylic acid selected from one or more of the group consisting of pyruvic acid, lactic acid, enolpyruvic acid, an amino acid, a fatty acid other than acetic acid, lipoic acid, nicotinic acid, pantothenic acid, succinic acid, fumaric acid, p-aminobenzoic acid, betahydroxybutyric acid, orotic acid, and carnitine, with the proviso that at least one R is not hydrogen, or the pharmaceutically acceptable salt thereof.
Broadly, the acyl derivatives of 2xe2x80x2-deoxyguanosine are those having the formula 
wherein R is hydrogen or an acyl radical of a metabolite other than acetyl, with the proviso that at least one R is not hydrogen, or the pharmaceutically acceptable salt thereof.
The preferred acyl derivatives of 2xe2x80x2-deoxyguanosine are those having the formula 
wherein R is H or an acyl group derived from a carboxylic acid selected from one or more of the group consisting of pyruvic acid, lactic acid, enolpyruvic acid, an amino acid, a fatty acid other than acetic acid, lipoic acid, nicotinic acid, pantothenic acid, succinic acid, fumaric acid, p-aminobenzoic acid, betahydroxybutyric acid, orotic acid, and carnitine, with the proviso that at least one R is not hydrogen, or the pharmaceutically acceptable salt thereof.
Broadly, the acyl derivatives of 2xe2x80x2-deoxycytidine are those having the formula 
wherein R is hydrogen or an acyl radical of a metabolite other than acetyl, with the proviso that at least one R is not hydrogen, or the pharmaceutically acceptable salt thereof.
The preferred acyl derivatives of 2xe2x80x2-deoxycytidine are those having the formula 
wherein R is H or an acyl group derived from a carboxylic acid selected from one or more of the group consisting of pyruvic acid, lactic acid, enolpyruvic acid, an amino acid, a fatty acid other than acetic acid, lipoic acid, nicotinic acid, pantothenic acid, succinic acid, fumaric acid, p-aminobenzoic acid, betahydroxybutyric acid, orotic acid, and carnitine, with the proviso that at least one R is not hydrogen, or the pharmaceutically acceptable salt thereof.
Broadly, the acyl derivatives of 2xe2x80x2-deoxythymidine are those having the formula 
wherein R is hydrogen or an acyl radical of a metabolite other than a fatty acid having less than five carbon atoms, with the proviso that at least one R is not hydrogen, or the pharmaceutically acceptable salt thereof.
The preferred acyl derivatives of 2xe2x80x2-deoxythymidine are those having the formula 
wherein R is B or an acyl group derived from a carboxylic acid selected from one or more of the group consisting of pyruvic acid, lactic acid, enolpyruvic acid, an amino acid, a fatty acid containing 5 or more carbon atoms, lipoic acid, nicotinic acid, pantothenic acid, succinic acid, fumaric acid, p-aminobenzoic acid, betahydroxybutyric acid, orotic acid and carnitine, with the proviso that at least one R substituent is not hydrogen, or the pharmaceutically acceptable salt thereof.
The acyl derivatives of 2xe2x80x2-deoxythymidine may also be those having the formula 
wherein Rxe2x80x3 is hydrogen or an acyl radical of a metabolite, with the proviso that the Rxe2x80x3 on nitrogen is not hydrogen, or the pharmaceutically acceptable salt thereof.
Preferred acyl derivatives of 2xe2x80x2-deoxythymidine are those having the formula 
wherein Rxe2x80x3 is H or an acyl group derived from a carboxylic acid selected from one or more of the group consisting of pyruvic acid, lactic acid, enolpyruvic acid, an amino acid, a fatty acid, lipoic acid, nicotinic acid, pantothenic acid, succinic acid, fumaric acid, p-aminobenzoic acid, betahydroxybutyric acid, orotic acid, and carnitine, with the proviso that the Rxe2x80x3 on nitrogen is not hydrogen, or the pharmaceutically salt thereof.
The invention also includes compounds having formulae I-IV wherein the ribose moiety is monoacylated at the 3xe2x80x2 or 5xe2x80x2 position with the derivative of a fatty acid and includes 3xe2x80x2,5xe2x80x2 diacylated derivatives of compounds I-IV wherein at least one such substituent is derived from a fatty acid having 5 or more carbon atoms.
The acyl derivatives of 2xe2x80x2-deoxyadenosine, 2xe2x80x2-deoxyguanosine, 2xe2x80x2-deoxycytidine, and 2xe2x80x2-deoxythymidine having formulae I, II, III, and V, desirably are substituted with an acyl derivative of a carboxylic acid having 3-22 carbon atoms.
Where acyl derivatives of any of the compounds of formulae I-V are substituted by an acyl group derived from an amino acid, the amino acid is desirably selected from the group consisting of glycine, the L forms of alanine, valine, leucine, isoleucine, phenylalanine, tyrosine, proline, hydroxyproline, serine, threonine, cysteine, cystine, methionine, tryptophan, aspartic acid, glutamic acid, arginine, lysine, histidine, ornithine, and hydroxylysine.
In a preferred embodiment of the invention, a mixture of at least two acyl derivatives of 2xe2x80x2-deoxyadenosine, 2xe2x80x2-deoxyguanosine, 2xe2x80x2-deoxycytidine, and 2xe2x80x2-deoxythymidine is used. Said compositions contain at least two of the acyl derivatives having the formulae 
wherein Rxe2x80x2xe2x80x3 is H or an acyl group derived from a carboxylic acid selected from one or more of the group consisting of pyruvic acid, lactic acid, enolpyruvic acid, an amino acid, a fatty acid, lipoic acid, nicotinic acid, pantothenic acid, succinic acid, fumaric acid, p-aminobenzoic acid, betahydroxybutyric acid, orotic acid, and carnitine, with the proviso that at least one R is not hydrogen, or the pharmaceutically acceptable salt thereof.
Further substantial benefits may be obtained, particularly where the compositions of the invention are used to ameliorate the effects of radiation, if a radioprotective compound is included together with one or more of the acyl deoxyribonucleosides. The radioprotective compounds may be those selected from the group consisting of WR-2721, NAC, DDC, cysteamine, 2-mercaptoethanol, mercaptoethylamine dithiothreitol, glutathione, 2-mercaptoethanesulfonic acid, WR-1065, nicotinamine, 5-hydroxytryptamine, 2-aminoethyl-isothiouronium-Brxe2x80x94Hbr, glucans, GLP/B04, GLP/B05, OK-432, Biostim, PSK, Lentinan, Schizophyllan, Rhodexman, Levan, Mannozym, MVE-2, MNR, MMZ, IL-1, TNF, thymic factor TF-5, glutathione peroxidase, superoxide dismutase, catalase, glutathione reductase, glutathione transferase, selenium, CdCl2, MnCl2, Zn acetate, Vitamin A, beta carotene, prostaglandins, tocopherol, methylene blue and PABA.
The invention is also embodied in pharmaceutical compositions which comprise one or more of the novel deoxyribonucleosides together with a pharmaceutically acceptable carrier. In addition, known acetyl derivatives of the 2xe2x80x2-deoxyadenosine, 2xe2x80x2-deoxyguanosine, 2xe2x80x2-deoxycytidine and 2xe2x80x2-deoxythymidine as well as the fatty acid derivatives of thymidine wherein the acyl group contains 3 or 4 carbon atoms may be used alone, in combination with one another or in combination with one or more novel compounds, in pharmaceutical compositions of the invention. The composition may further include a radioprotective compound as described. The compositions may be in the form of a liquid, a suspension, a tablet, a dragee, an injectable solution, a topical solution, or a suppository.
A skin lotion may be advantageously prepared by combining an effective amount of one or more of the acyl deoxyribonucleosides of the invention together with a suitable carrier. Such a skin lotion advantageously contains from 0.1 to 5 percent by weight of the deoxyribonucleosides and, if desirable, the radioprotective compound.
The pharmaceutical compositions of the invention can also be embodied in bioerodible microcapsules, the microcapsules desirably being selected from the group consisting of polylactate or lactate-glycolate copolymers.
It is believed that the delivery of exogenous deoxyribonucleosides to the tissue of an animal can be effectively achieved by administering to that animal an effective amount of an acyl derivative of a deoxyribonucleoside of formulae I-V. By enhancing the delivery of exogenous deoxyribonucleosides, and thereby increasing their bioavailability, it may be possible to treat physiological or pathological conditions of the tissues of an animal by essentially supporting the metabolic functions thereof. Without being bound by theory, the invention may work, as well, by increasing the bioavailability of nucleoside anabolites e.g. nucleotides or nucleotide-derived cofactors. Administration of the nucleosides per se increases their bioavailability but, due to rapid catabolism, this may not result in significant elevation of nucleotide levels; i.e., one does not necessarily get an increase in intracellular levels because at lower nucleoside levels there is rapid uptake by the cells whereas at higher levels there is saturation and the excess is degraded. The invention is believed to work by delivering a steady supply of nucleoside at low levels.
It is believed that the novel compounds and compositions of the invention may be used advantageously in methods for treating cardiac insufficiency, myocardial infarction, the consequences of hypertension, cirrhosis of the liver, diabetes, senescence, adrenal insufficiency, the complications of pregnancy, cerebrovascular disorders, senile dementias, Parkinson""s disease, demyelinating disorders, cerebellar ataxia, infant respiratory distress syndrome, and lung disorders, or to enhance bone healing or muscle performance.
The specific methods where advantages may be achieved using the compounds and compositions of the invention include treating or preventing radiation-induced cellular damage, preventing sunlight-induced cellular damage, ameliorating the effects of aging, preventing mutagen-induced cellular damage, healing damaged tissue, healing skin wounds, healing burn tissue, healing diseased or damaged liver tissue, healing heart muscle damaged as a result of myocardial infarction, treating damaged bone marrow, and enhancing erythropoiesis. In treating all of these conditions, a compound of the invention, with or without additional carriers, radioprotective compounds, and other adjuvants, are administered to an animal.
The invention essentially enhances the transport of deoxyribonucleosides across biological membranes, including the gastrointestinal tract (i.e., transport from the gut into the bloodstream) and the blood-brain barrier. The rapid catabolism by nucleoside phosphorylases or nucleoside deaminases is also substantially prevented.
Administration of the acylated derivatives offers certain advantages over the nonderivatized compounds. The acyl substituents can be selected to increase the lipophilicity of the nucleoside, thus improving its transport from the gastrointestinal tract into the bloodstream. The acylated derivatives are effective when administered orally. The acylated derivatives are resistant to catabolism by nucleoside deaminases and nucleoside phosphorylases in the intestine, liver, other organs, and the bloodstream. Thus, administration of the acylated derivatives of the invention, either orally or parenterally, allows sustained delivery of high levels of deoxyribonucleosides to the tissues of an animal.